Abstract

Introduction

Chondrogenesis is a well-orchestrated, multicellular process driven by chondrogenic progenitors that undergo mesenchymal condensation, proliferation, chondrocyte differentiation and maturation [1, 2]. The environment in which the chondro-progenitors reside during this process is crucial in establishing chondrogenesis in vitro [3, 4]. Studies using bone marrow-derived stromal cells (BMSCs) and articular chondrocytes have achieved chondrogenesis in three-dimensional (3D) culture using either a micromass technique, pellet culture, agarose suspension culture, or by seeding cells into a matrix platform such as alginate beads or a PLGA scaffold [5-10]. Most recently we have demonstrated that ambient oxygen is a critical micro-enviromental regulator during chondrogenic differentiation and influences the fate of chondrocyte [11].

Adipose-derived stromal cells (ASC) are a promising cell source for tissue engineering because of their abundant supply and their capacity for differentiating to bone, cartilage, muscle and fat [12-16]. ASC are readily accessible, easily expandable and have low donor site morbidity; therefore, applying ASC as an alternative cell source for cartilage repair is an area of intense interest [17-19]. Most of our current research is focusing on studying the chondrogenic and osteogenic capacities of these cells [11, 16, 20-23]. Because of the low proliferation capability of explanted chondrocytes our group and others are investigating several in vitro chondrogenesis differentiation systems using ASC.

Recently, the nanomechanical properties (e.g. hardness and elastic modulus) have been used to measure the material properties of calcified biological tissues [24, 25]. Therefore, nanoindentation as a high-resolution probe has received a great deal of attention and provided a method for determining material properties on a highly reduced scale [25, 26]. With this technique, direct physical property measurements of heterogeneous biological materials, such as friction, adhesion and elasticity, can be determined with high spatial resolution. In addition, the time-dependent mechanical properties of these materials can be obtained in aqueous environments, providing a means for property determination that approximates the in situ tissue environment. In particular, cartilage behaves mechanically as a viscoelastic solid due to its composition and structure [27-29]. The mechanical properties of cartilage, such as elastic modulus, depend on the rate of strains which are controlled by the amount of extracelluar matrix (ECM) formation, collagen deposition and degradation.

In this study, we utilized a 3D micromass culture system of ASC to create a structural micro-environment for these cells undergoing early chondrogenic differentiation. Subsequently, we have observed the significant differentiation dynamics through the material properties of the differentiated micromasses. We conclude that this in vitro, 3D micromass system will be useful for elucidating the mechanism(s) underlying early chondrocyte differentiation and that nanoindentation is a powerful tool to evaluate the material properties of cartilage/chondrocytes during early chondrogenic differentiation.

Materials and Methods

In vitro Chondrogenic Experimental Design

The micromass technique was modified from Ahrens et al. [4, 11, 20]. Briefly, passage two cells were trypsinized and resuspended in growth media at a density of 100,000 cells/10μl. Ten-microliter droplets were seeded in culture dishes and allowed to form cell aggregates and substratum at 37°C for two and half hours. Chondrogenic media (containing DMEM, 1% FBS, 1% penicillin/streptomycin, 37.5 μg/ml ascorbate-2-phophate, ITS premix, and 10ng/ml TGF-β1) was carefully added around the cell aggregates. The chondrogenic media was replenished every three days (Figure 1).

Open in a separate window

Figure 1

Schematic process of experimental design for 3D micromasses creation

1A) Schematic for cell harvest and high density seeding technique: mouse inguinal fat pads were harvested, digested, and pelleted. Cells were resuspended in culture media at a density of 100,000 cells/10 μl; 10 μl droplets were placed in tissue culture dishes and allowed to dry for two hours.

1B) Schematic for micromass formation: chondrogenic media containing TGF-β1 at 10ng/ml was added to the cell aggregates. Aggregates condensed and formed spheroids within 24 hours of chondrogenic media addition.

1C) Microscopic view of cell aggregates and condensation of 3D micromasses. The micromasses are shown post-seeding from 12 hour to 96 hours of condensation and aggregation in chondrogenic culture (10x magnification).

Material Properties of Differentiated Micromasses

In the present experiments, the conventional atomic force microscope (AFM) head was replaced by an electrostatic operated transducer that allowed for topographic imaging as well as local indentation capability (TriboScope nanoindenter, Hysitron, Minneapolis, MN, USA) and mounted on a multimode AFM controlled by NanoScope IIIa electronics (Veeco, Santa Barbara, CA). In the indentation mode, the electrostatic force acting on the spring-suspended center plate of the force—displacement transducer allows the detecting of the time dependence force behavior for prescribed displacement. The detailed description of the instruments has been previously published [25, 26]. All experiments were performed in a fluid cell encapsulating samples. A quartz sphere, glued to tungsten rode that is connected to the middle plate of the transducer (Figure 2A and 2B) acts as an indenter. The samples, sphere and portion of the tungsten rod were immersed in water during the experiments.

Open in a separate window

Figure 2

Nanoindentation of the differentiated micromasses

2A) Schematic of indentation technique to measure material properties of micromasses.

2B) A digital picture of the glass bead attached to the diamond tip.

2C) Theoretical load displacement curves from micromasses. Displacement is shown in red, load is shown in black.

2D) Experimental load vs. time curves indicating the error associated with the modulus calculations.

The typical force-displacement curve for cartilage sample is shown in Figure 3A using a 164 μm sphere radius. The results were analyzed based on Kelvin model:

$$F\left(t\right)=S\left[1-\left(1-\frac{{\tau }_{\sigma }}{{\tau }_{\epsilon }}\right)\cdot \exp \left(-\frac{t}{{\tau }_{\epsilon }}\right)\right]\cdot d\left(t\right)$$

(1)

where F(t) , d(t), S, and t are applied force, displacement, stiffness, and time, respectively. The moduli are calculated from Hertz formula for sphere with radius R:

$$E_S^{\text{Hertz}}=\sqrt{\frac{S^3{(1-V{s}^2)}^2}{6RF}}$$

(2)

where Poisson’s ratio, ν, is assumed 0.5.

Open in a separate window

Figure 3

Changes of material properties during micromass differentiation

3A) The theoretical load displacement curve used to determine the relaxed modulus of the micromasses. The force (red) and displacement (blue) are used to determine the stiffness.

3B) The dynamic modulus, and 3C) the relaxed modulus of micromasses at days 3, 6, 9, 12, and 15. A significant increase in dynamic modulus (*p<.001) is seen at day 15 compare to day 3. The relaxation moduli values showed significant differences at days 6, 9, 12, and 15 compared to day 3 relaxed modulus (*p<.001).

Results

Discussion

Articular cartilage in adults has limited capacity for self-repair following injury. Autologous chondrocyte transplantation is a cell-based orthobiologic technology used for the treatment of cartilage defects [30]. Surgical therapeutic efforts to treat cartilage defects have focused on delivering new, in vitro-cultured cells capable of chondrogenesis into the lesions [31]. However, the poor proliferative potential of the chondrocytes in vitro has been a major obstacle for stem cell research and the development of clinical therapies. Much of the current research is focused on developing strategies to improve chondrocyte culture expansion and selection [20, 31]. Therefore, using ASC, a readily available and abundant potential source of mesenchymal stem cells is a promising approach for cartilage repair/regeneration.

The influence of mechanical stimuli within micro-environments has been extensively described as a large contributor to chondrogenic differentiation in vitro. Chondrocytes appear to maintain their phenotype in a 3D culture [5, 32]. Consistent with previous reports, we have achieved successful early chondrogenic differentiation in ASC by using the 3D micromass culture system [11, 20]. Alcian blue and collagen II staining indicated the progression of chondrogenesis in the 3D micromasses. In addition to the histology, we have assessed sulfated glycosaminoglycan (sGAG) quantities to evaluate this micromass culture system [33]. Gene expression data demonstrated that ASC underwent early chondrogenesis and obtained the characteristics of prechondrocytes: such as significantly higher levels of Sox-9, the critical transcription factor of chondrogenesis; elevated aggrecan and type II collagen, major components of chondrogenesis [11, 20]. Collectively, these analyses provide details of spatial-temporal expression of molecules regulating chondrogenic differentiation in ASC micromasses.

Building on our previous reported changes in histology and marker genes, in this study we also observed significant changes in material properties of the differentiated micromasses. Lacking the technological capabilities of high spatial resolution, analysis of the mechanical properties of differentiated mesenchymal stem cells is still in its infancy. However, in this study, by using a spherical glass bead tip in AFM nanoindentation, we were able to increase the surface area while not compromising the high spatial resolution of the nanoindentation technique. Furthermore, the use of nanoindentation allowed us to assess the properties of these micromasses in nano-scale measurements. Therefore, a significant increase of mechanical integrity was observed over the time of differentiation. Our data indicated that the surrounding micro-environmental factors combined with the 3D structure provide appropriate stimuli and create a niche for early ASC chondrogenesis.

ASC are a heterogeneous cell population consisting of cells in various stages of differentiation and levels of potency. Defining which cells within this population differentiate along specific lineages is a central goal of our research. Factors involved in the regulation of early chondrogenesis will be further determined by utilizing the in vitro 3D chondrogenic system. Novel methods of priming and selecting for chondro-progenitors within the heterogeneous population of ASC will contribute to translational therapies utilizing ASC for cartilage repair/replacement. In conclusion, we report that a 3D in vitro micromass system represents a dynamic multi-cellular model of early chondrogenesis associated with the mechanical properties of early chondrocyte differentiation.

Acknowledgements

Footnotes

References